**4. Indirect immunofluorescence tecnique**

Third generation fibroblast cell cultures were exposed to the different mixtures of contaminants reported in the Table 1.

We used immunofluorescence in fibroblast cultures for a qualitative and semi-quantitative analysis of target proteins CYP1A1, CYP2B and MICA. After a first reaction with the primary polyclonal antibodies (goat anti-rabbit cytochrome P450 1A1 and goat anti-rabbit cytochrome P450 2B; Oxford Biochemical Research (Oxford MI, USA); rabbit polyclonal anti-MICA; Abcam), the cells were treated with the respective secondary antibodies (Alexa Fluor 594 goat anti-rabbit IgG (H+L) for CYP1A1 and CYP2B; Alexa Fluor 568 rabbit anti-goat IgG (H+L) for MICA; Invitrogen), labelled with red-fluorescent Alexa Fluor dye. Immunofluorescence was quantified with a specially designed Olympus Soft Imaging Systems macro, *DetectIntZ,* which works with the image acquisition, processing and analysis system, *analySIS^B* (Olympus) [15]. The image analysis procedure has the objective of quantifying, with an adimensional index generated for this purpose, the amount of Alexa

"Test Tube Cetaceans": From the Evaluation of Susceptibility to the Study of Genotoxic Effects of Different Environmental Contaminants Using Cetacean Fibroblast Cell Cultures 53


**Table 1.** The different mixtures of contaminants and doses to which cell cultures were exposed.

Fluor localized in the membrane of cytoplasmatic area of sample cells. The sample cells are imaged using DAPI and this image is presented to the operator for threshold selection of cytoplasmatic and nuclei Region of Interests (ROIs) across the field. The procedure then utilizes these ROIs to measure fluorescence intensity of Alexa Fluor sample cell and summarizes the results in a worksheet. The system generates index values which are unitless until compared with other units, such as number of cells to obtain mean fluorescence per cell or the area in which it is calculated to obtain mean fluorescence per mm2. Images are all obtained with a magnification of 20X, a calibration of 0.65 μm/pixel and a resolution of 1360 x 1024 x 8 pixel. Exposure times were maintained fixed while reading the CYP1A1, CYP2B and MICA for each treatment. A series of images of each slide was acquired so that a minimum of 250 cells/slide could be counted. The total fluorescence revealed by the program is divided by number of cells to obtain arbitrary unity of fluorescence (AUF) per cell. Several slides for CYP1A1, CYP2B and MICA were made for each culture: one was a blank (cells treated only with primary and secondary antibodies), one was a secondary blank (cells treated only with secondary antibody), one was a chemical blank (cells treated with contaminant carrier), two were for each treatment dose of contaminants. The blank enabled the natural presence of the target proteins in cultured fibroblasts to be checked. The secondary blank enabled validation of the dose of secondary antibody without cross reaction as the primary antibody was absent.
